Polysubstituted thiazolylpyridine carboxyamide antifungal antibiotic

ABSTRACT

Novel antifungal antibiotic compounds have the structural formula &lt;IMAGE&gt;   A preferred compound where R is 4-hydroxypentyl is denoted BU-3557B2. A complex of said compounds is produced by fermenting a culture of Sacchrothrix aerocolonigenes strain N806-4 (ATCC 53712). The complex is recovered by adsorption on nonionic porous polymer resin adsorbent and individual component compounds exhibit in vitro activity against fungi and Gram-positive bacteria and BU-3557B2 demonstrates in vitro antiprotazoal activity. BU-3557B2 demonstrates in vivo activity against C. albicans vaginal infection and BU-3557A3 (where R is 1,5-dihydroxy-5,5-dimethylpentyl) demonstrates in vivo activity against C. albicans systemic infection.

FIELD OF THE INVENTION

This invention relates to novel antifungal antibiotic compounds, theirproduction, and their use in treating fungal infections. This inventionalso relates to a novel culture for use in producing antifungalantibiotic.

BACKGROUND OF THE INVENTION

There are relatively few known effective antifungal antibiotics. Becauseof this, there has been a continuing search to uncover microorganismsfor screening for production of antifungal fermentation products. Thepresent invention is the result of this effort.

SUMMARY OF THE INVENTION

The novel antifungal antibiotic compounds herein have the followingstructural formula: ##STR2## wherein R is pentyl having one or moresubstituents at one or more positions selected from the group consistingof lower alkyl (i.e. C₁ -C₃ alkyl), hydroxy and oxo.

A preferred compound is denoted BU-3557B₂ and has the above structuralformula where R is 4-hydroxypentyl.

A complex of said compounds is produced by fermenting a culture of anewly uncovered strain of Saccharothrix designated Saccharothrixaerocolonigenes strain N806-4 (ATCC 53712). The production methodinvolves cultivating said strain under aerobic conditions in an aqueousnutrient medium containing assimilable sources of carbon and nitrogenuntil a substantial amount of said complex is produced and recoveringsaid complex. Individual compounds can be recovered from said complex bychromatography.

A pharmaceutical composition for treatment of antifungal infectiouscomprises and effective antifungal amount of compound as described abovein combination with pharmaceutical carrier of diluent.

An animal host affected by a fungal infection sensitive to compound ofthe present invention is therapeutically treated by administering tosaid host an effective antifungal dose of said compound in the form ofsaid pharmaceutical composition. In the therapeutic treatment of Candidaalbicans systemic infection, administration of compound of the presentinvention is readily carried out by topical application.

DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the infrared spectrum of BU-3557 A₃, i.e., compound ofthe above structural formula where R is1,5-dihydroxy-5,5-dimethylpentyl.

FIG. 2 depicts the ¹ H-NMR spectrum of BU-3557 A₃ at 400 MHz, in CDCl₃.

FIG. 3 depicts the infrared spectrum of BU-3557B₂, i.e., compound of theabove structural formula where R is 4-hydroxypentyl.

FIG. 4 depicts the ¹ H-NMR spectrum of BU-3557B₂ at 400 MHz in CDCl₃.

FIG. 5 depicts the infrared spectrum of BU-3557C₄, i.e., compound of theabove structural formula where R is 3-methyl-4-oxopentyl.

FIG. 6 depicts the ¹ H-NMR spectrum of BU-3557C₄ at 400 MHz in CDCl₃.

FIG. 7 depicts the infrared spectrum of BU-3557D, i.e., compound of theabove structural formula where R is 5-hydroxy-5,5-dimethylpentyl.

FIG. 8 depicts the ¹ H-NMR spectrum of BU-3557D at 400 MHz in CDCl₃.

DETAILED DESCRIPTION

In the novel compounds herein the substituents on the pentyl of Rtypically are 1 to 4 in number and occur at 1 or positions especially atthe 1-position, 3-position, 4-position and/or 5-position and areselected from the group consisting of methyl, hydroxy and oxo. In 7compounds that have been prepared R contains an oxo substituent or ahydroxy substituent at the 4-position of its pentyl.

Specific compounds that have been prepared and identified include thosewith the above structural formula where R is 1,4-dihydroxypentyl(designated BU-3557A₁), where R is 1,4-dihydroxy-4-methylpentyl(designated BU-3557A₂), where R is 1,5-dihydroxy-5,5-dimethylpentyl(designated BU-3557A₃), where R is 4-oxopentyl (designated BU-3557B₁),where R is 4-hydroxypentyl (designated BU-3557B₂), where R is4-hydroxy-4-methylpentyl (designated BU-3557B₃), where R is a3-hydroxypentyl (designated BU-3557C₁), where R is4-methyl-5-hydroxypentyl (designated BU-3557C₂), where R is3-methyl-4-hydroxypentyl (designated BU-3557C₃), where R is3-methyl-4-oxopentyl (designated BU-3557C₄), where R is 1-hydroxypentyl(designated BU-3557C₅), and where R is 5-hydroxy-5,5-dimethylpentyl(designated BU-3557D). BU-3557B₂ is the preferred compound herein inthat it has been found to be relatively active. BU-3557B₂ has beendetermined by x-ray diffraction to be6-[2-(4-hydroxypentyl)thiazol4-yl]-3-hydroxy-4,5-dimethoxypyridine-2-carboxamide.

Physico-chemical properties for each of the specific compounds referredto above are set forth in Table I below:

                                      TABLE I                                     __________________________________________________________________________                BU-3557A.sub.1                                                                           BU-3557A.sub.2                                                                           BU-3557A.sub.3                                                                            BU-3557B.sub.1                  __________________________________________________________________________    Nature      White powder                                                                             White powder                                                                             White powder                                                                              White crystalline                                                             powder                          M.p.        56.5-58.5° C.                                                                     60-63° C.                                                                         69-72° C.                                                                          151-153° C.              [α].sub.D.sup.25                                                                    -11°(c = 0.5, CHCl.sub.3)                                                         -14°(c = 0.5, CHCl.sub.3)                                                         -19°(c = 0.25,                                                                     0°(c = 0.5,                                                            CHCl.sub.3)                     UV λ.sub.max (ε) nm                                            in MeOH     219(25,000)                                                                              219(25,500)                                                                              218(26,400) 219(25,900)                                 242(22,700)                                                                              242(23,500)                                                                              242(23,700) 242(23,600)                                 315(6,700) 316(6,800) 317(6,800)  316(6,900)                      in 0.1N HCl--MeOH                                                                         220(25,700)                                                                              220(23,900)                                                                              220(25,000) 220(23,900,sh)                  (1:9)       243(25,700)                                                                              243(23,900)                                                                              243(25,100) 243(26,400)                                 318(7,500) 318(6,900) 317(7,300)  318(8,000)                      in 0.1N NaOH--MeOH                                                                        220(23,400)                                                                              220(23,400)                                                                              220(24,200) 220(23,300)                     (1:9)       251(21,600)                                                                              251(21,500)                                                                              251(22,200) 251(23,300)                                 281(17,200)                                                                              282(17,000)                                                                              282(17,400) 292(16,600)                                 342(9,000) 342(9,000) 342(9,200)  341(9,100)                      IR ν.sup.KBr .sub.max cm.sup.-1                                                        3400,1665,1590                                                                           3300,1665,1580                                                                           3350,1665,1580                                                                            3385,1705,1665,1600             MS (EI) m/z 383(M.sup.+),368,338                                                                     397(M.sup.+),382,364,323                                                                 411(M.sup.+),396,393,378                                                                  365(M.sup.+),295,219            Molecular formula                                                                         C.sub.16 H.sub.21 N.sub.3 SO.sub.6                                                       C.sub.17 H.sub.23 N.sub.3 SO.sub.6                                                       C.sub.18 H.sub.25 N.sub.3 SO.sub.6                                                        C.sub.16 H.sub.19 N.sub.3                                                     SO.sub.5                        __________________________________________________________________________                BU-3557B.sub.2                                                                           BU-3557B.sub.3                                                                          BU-3557C.sub.1                                                                            BU-3557C.sub.2                   __________________________________________________________________________    Nature      White crystalline                                                                        White crystalline                                                                       White crystalline                                                                         White crystalline                            powder     powder    powder      powder                           M.p.        171-172.5° C.                                                                     187.5-189.5° C.                                                                  152.5-154° C.                                                                      130.5-133° C.             [α].sub.D.sup.25                                                                    0°(c = 0.5, CHCl.sub.3)                                                           0°(c = 0.5, CHCl.sub.3)                                                          +14°(c = 0.25, CHCl.sub.3)                                                         -8°(c = 0.25,                                                          CHCl.sub.3)                      UV λ.sub.max (ε) nm                                            in MeOH     219(26,100)                                                                              220(25,400)                                                                             220(25,000) 220(26,000)                                  242(23,500)                                                                              242(23,200)                                                                             243(22,200) 243(24,000)                                  317(6,800) 317(6,800)                                                                              317(6,600)  318(7,100)                       in 0.1N HCl--MeOH                                                                         220(23,500,sh)                                                                           220(22,100,sh)                                                                          222(23,100,sh)                                                                            222(22,500,sh)                   (1:9)       243(26,900)                                                                              243(26,500)                                                                             242(23,500) 243(26,600)                                  318(8,100) 318(7,900)                                                                              317(7,500)  318(8,200)                       in 0.1N NaOH--MeOH                                                                        221(23,300)                                                                              218(21,400)                                                                             220(22,100) 217(22,100)                      (1:9)       252(21,100)                                                                              252(20,200)                                                                             252(19,800) 253(20,600)                                  293(16,600)                                                                              294(16,200)                                                                             294(15,700) 294(16,800)                                  342(9,000) 342(8,700)                                                                              342(8,400)  342(9,100)                       IR ν.sup.KBr .sub.max cm.sup.-1                                                        3300,1675,1600                                                                           3240,1680,1610                                                                          3300,1670,1600                                                                            3200,1670,1600                   MS (EI) m/z 367(M.sup. +),352,335                                                                    381(M.sup.+),366,332                                                                    367(M.sup.+),352,338                                                                      381(M.sup.+),366,295             Molecular formula                                                                         C.sub.16 H.sub.21 N.sub.3 SO.sub.5                                                       C.sub.17 H.sub.23 N.sub.3 SO.sub.5                                                      C.sub.16 H.sub.21 N.sub.3 SO.sub.5                                                        C.sub.17 H.sub.23 N.sub.3                                                     SO.sub.5                         __________________________________________________________________________                BU-3557C.sub.3                                                                            BU-3557C.sub.4                                                                           BU-3557C.sub.5                                                                            BU-3557D                       __________________________________________________________________________    Nature      White crystalline                                                                         White crystalline                                                                        White crystalline                                                                         White crystalline                          powder      powder     powder      powder                         M.p.        165.5-167.5° C.                                                                    124.5-126.5° C.                                                                   138-140° C.                                                                        146.5-148° C.           [α].sub.D.sup.25                                                                    +20°(c = 1.0, CHCl.sub.3)                                                          0°(c = 1.0, CHCl.sub.3)                                                           +19°(c = 1.0,                                                                      0°(c = 1.0,                                                            CHCl.sub.3)                    UV λ.sub.max (ε) nm                                            in MeOH     220(25,800) 220(25,900)                                                                              220(25,700) 219(26,300)                                242(23,200) 243(23,900)                                                                              243(23,900) 242(23,500)                                317(6,800)  317(7,100) 317(7,100)  317(6,800)                     in 0.1N HCl--MeOH                                                                         222(25,000,sh)                                                                            223(23,200,sh)                                                                           220(24,600) 220(23,300,sh)                 (1:9)       244(28,300) 243(26,100)                                                                              243(24,700) 243(26,400)                                318(8,400)  318(8,000) 317(7,400)  317(8,000)                     in 0.1N NaOH--MeOH                                                                        220(17,300) 217(22,300)                                                                              217(22,200) 221(22,900)                    (1:9)       251(21,000) 252(20,600)                                                                              252(20,700) 252(20,800)                                294(16,600) 294(16,700)                                                                              283(16,700) 292(16,400)                                341(8,800)  342(9,100) 343(8,900)  342(8,800)                     IR ν.sup.KBr .sub.max cm.sup.-1                                                        3200,1670,1605                                                                            3390,1705,1660,1590                                                                      3300,1670,1600                                                                            3250,1675,1610                 MS (EI) m/z 381(M.sup.+),366,336                                                                      379(M.sup.+),364,308                                                                     367(M.sup.+),352,311                                                                      395(M.sup.+),380,362           Molecular formula                                                                         C.sub.17 H.sub.23 N.sub.3 SO.sub.5                                                        C.sub.17 H.sub.21 N.sub.3 SO.sub.5                                                       C.sub.16 H.sub.21 N.sub.3 SO.sub.5                                                        C.sub.18 H.sub.25 N.sub.3                                                     SO.sub.5                       __________________________________________________________________________

Analysis of ¹ H-NMR spectra for each of the compounds specificallymentioned above at (400 MHz, in CDCl₃) is set forth in Table II below.

                                      TABLE II                                    __________________________________________________________________________             BU-3557A.sub.1                                                                      BU-3557A.sub.2                                                                         BU-3557A.sub.3                                                                      BU-3557B.sub.1                                                                       BU-3557B.sub.2                                                                      BU-3557B.sub.3                     __________________________________________________________________________    Side chain ®                                                              C-1      5.17(1H,t)                                                                          5.33(1H,t)                                                                             5.38(1H,t)                                                                          3.14(2H,t)                                                                           3.00(1H,m)                                                                          3.06(2H,t)                                                              3.25(1H,m)                               2        2.12(2H,m)                                                                          2.13(1H,m)                                                                             2.04(2H,m)                                                                          2.12(2H,qui)                                                                         2.00(1H,m)                                                                          2.00(2H,qui)                                      2.24(1H,m)            2.20(1H,m)                                3        1.68(2H,m)                                                                          1.78(2H,m)     2.59(2H,t)                                                                           1.64(2H,m)                                                                          1.65(2H,t)                                                1.61(4H,m)                                            4        3.90(1H,m)                  3.94(1H,m)                               5        1.22(3H,t)                                                                          1.26(3H,s)     2.15(3H,s)                                                                           1.18(3H,d)                                                                          1.13(3H,s)                         6              1.26(3H,s)                                                                             1.19(3H,s)         1.13(3H,s)                         7                       1.22(3H,s)                                            Aromatic nucleus                                                              --OCH.sub.3                                                                            3.95(3H,s)                                                                          4.01(3H,s)                                                                             4.00(3H,s)                                                                          3.96(3H,s)                                                                           3.97(3H,s)                                                                          3.89(3H,s)                                  4.06(3H,s)                                                                          4.11(3H,s)                                                                             4.11(3H,s)                                                                          4.12(3H,s)                                                                           4.09(3H,s)                                                                          3.94(3H,s)                         Aromatic --H                                                                           7.88(1H,s)                                                                          7.98(1H,s)                                                                             7.98(1H,s)                                                                          7.81(1H,s)                                                                           7.90(1H,s)                                                                          7.83(1H,s)                         --NH or  5.80(1H,br)                                                                         5.70(1H,br)                                                                            5.76(1H,br)                                                                         5.84(1H,br)                                                                          5.62(1H,br)                                                                         5.53(1H,br)                        --OH     9.15(1H,br)                                                                         9.32(1H,br)                                                                            9.40(1H,br)                                                                         8.43(1H,br)                                                                          6.88(1H,br)                                                                         5.70(1H,br)                                 12.65(1H,s)                                                                         12.88(1H,s)                                                                            12.93(1H,s)                                                                         12.49(1H,s)                                                                          9.78(1H,br)                                                                         9.69(1H,br)                                                             12.91(1H,s)                                                                         12.81(1H,s)                        __________________________________________________________________________             BU-3557C.sub.1                                                                        BU-3557C.sub.2                                                                      BU-3557C.sub.3                                                                      BU-3557C.sub.4                                                                        BU-3557C.sub.5                                                                        BU-3557D                         __________________________________________________________________________    Side chain ®                                                              C-1      3.25(2H,m)                                                                            3.25(2H,t)                                                                          3.15(2H,t)                                                                          3.07(2H,t)                                                                            5.22(1H,t)                                                                            3.20(2H,t)                       2        2.05(2H,m)    2.13(2H,m)                                                                          1.86(1H,m)                                                                            1.95(2H,m)                                                                            1.90(2H,tt)                                                   2.24(1H,m)                                        3        3.75(1H,m)                                                                            1.3-2.1                                                                             1.70(1H,m)                                                                          2.65(1H,qt)                                                                           1.2-1.6                                                                              1.57(4H,m)                       4        1.52(2H,m)                                                                            (5H,m)                                                                              3.55(1H,m)    (4H,m)                                   5        0.98(3H,t)                                                                            3.55(2H,t)                                                                          1.18(3H,d)                                                                          2.16(3H,s)                                                                            0.94(3H,t)                               6                0.92(3H,d)                                                                          0.95(3H,d)                                                                          1.17(3H,d)      1.19(3H,s)                       7                                            1.19(3H,s)                       Aromatic nucleus                                                              --OCH.sub.3                                                                            4.00(3H,s)                                                                            3.96(3H,s)                                                                          3.98(3H,s)                                                                          3.94(3H,s)                                                                            3.92(3H,s)                                                                            3.97(3H,s)                                4.10(3H,s)                                                                            4.10(3H,s)                                                                          4.08(3H,s)                                                                          4.11(3H,s)                                                                            4.10(3H,s)                                                                            4.11(3H,s)                       aromatic --H                                                                           7.80(1H,s)                                                                            7.92(1H,s)                                                                          7.90(1H,s)                                                                          7.77(1H,s)      7.90(1H,s)                       --NH or  5.58(1H,br)                                                                           5.60(1H,br)                                                                         5.55(1H,br)                                                                         5.89(1H,br)                                                                           5.95(1H,br)                                                                           5.65(1H,br)                      --OH     8.98(1H,br)                                                                           9.70(1H,br)                                                                         9.85(1H,br)                                                                         8.25(1H,br)                                                                           6.70(1H,br)                                                                           9.39(1H,br)                               12.65(1H,s)                                                                           12.90(1H,s)                                                                         12.90(1H,s)                                                                         12.41(1H,s)                                                                           9.30(1H,br)                                                                           12.78(1H,s)                                                           12.97(1H,s)                              __________________________________________________________________________

The ¹ H-NMR spectra for BU-3557A₃, BU-3557B₂, BU-3557Chd and BU-3557Dare respectively presented in FIGS. 2, 4, 6 and 8.

Analysis of the ¹³ C-NMR spectra for compounds BU-3557A₃, BU-3557B₁,BU-3557B₂, BU-3557B₃, BU-3557C.sub. 3, BU-3557C₄ and BU-3557D is setforth in Table III below.

                                      TABLE III                                   __________________________________________________________________________             BU-3557A.sub.3                                                                      BU-3557B.sub.1                                                                      BU-3557B.sub.2                                                                      BU-3557B.sub.3                                                                      BU-3557C.sub.3                                                                      BU-3557C.sub.4                                                                      BU-3557D                         __________________________________________________________________________    Side chain ®                                                              C-1      71.31                                                                             d 32.33                                                                             t 31.70                                                                             t 32.54                                                                             t 30.06                                                                             t 32.75                                                                             t 32.13                                                                             t                            2        38.16                                                                             t 23.86                                                                             t 22.44                                                                             t 21.07                                                                             t 28.11                                                                             t 30.84                                                                             t 29.30                                                                             t                            3        19.76                                                                             t 42.40                                                                             t 38.98                                                                             t 41.34                                                                             t 40.89                                                                             d 46.18                                                                             d 22.36                                                                             t                            4        42.55                                                                             t 208.03                                                                            s 65.19                                                                             d 70.53                                                                             s 70.35                                                                             d 212.01                                                                            s 41.75                                                                             t                            5        71.14                                                                             s 30.05                                                                             q 23.30                                                                             q 29.71                                                                             q 21.22                                                                             q 28.40                                                                             q 70.94                                                                             s                            6        29.02                                                                             q             29.71                                                                             q 16.81                                                                             q 16.71                                                                             q 29.30                                                                             q                            7        29.61                                                                             q                               29.30                                                                             q                            Aromatic nucleus                                                              --OCH.sub.3                                                                            60.79                                                                             q 60.83                                                                             q 60.72                                                                             q 60.74                                                                             q 60.67                                                                             q 60.79                                                                             q 60.74                                                                             q                                     60.85                                                                             q 61.07                                                                             q 60.75                                                                             q 60.74                                                                             q 60.71                                                                             q 60.87                                                                             q 60.87                                                                             q                            aromatic 119.40                                                                            d 118.54                                                                            d 118.44                                                                            d 118.55                                                                            d 118.36                                                                            d 118.61                                                                            d 118.57                                                                            d                            carbon   127.38                                                                            s 127.12                                                                            s 127.59                                                                            s 127.58                                                                            s 127.66                                                                            s 127.31                                                                            s 127.37                                                                            s                            and      136.87                                                                            s 137.43                                                                            s 136.68                                                                            s 136.96                                                                            s 136.64                                                                            s 137.19                                                                            s 137.53                                                                            s                            amide    147.74                                                                            s 147.88                                                                            s 147.66                                                                            s 147.71                                                                            s 147.66                                                                            s 147.73                                                                            s 147.79                                                                            s                            carbon   149.89                                                                            s 150.26                                                                            s 149.86                                                                            s 149.94                                                                            s 149.87                                                                            s 150.39                                                                            s 150.40                                                                            s                                     150.47                                                                            s 150.59                                                                            s 150.27                                                                            s 150.36                                                                            s 150.27                                                                            s 150.47                                                                            s 150.50                                                                            s                                     152.97                                                                            s 152.81                                                                            s 152.97                                                                            s 152.92                                                                            s 152.98                                                                            s 152.55                                                                            s 152.78                                                                            s                                     172.19                                                                            s 169.95                                                                            s 170.75                                                                            s 170.13                                                                            s 170.98                                                                            s 169.69                                                                            s 170.31                                                                            s                                     175.15                                                                            s 171.70                                                                            s 172.41                                                                            s 172.37                                                                            s 172.47                                                                            s 171.84                                                                            s 172.10                                                                            s                            __________________________________________________________________________

The IR spectra for BU-3557A₃, BU-3557B₂, BU-3557C₄ and BU-3557D arerespectively presented in FIGS. 1, 3, 5 and 7.

Each of the specific compounds referred to in Table I was soluble inbenzene, chloroform, dimethyl sulfoxide, ethyl acetate, methanol andethanol, but insoluble in n-hexane and water. They gave positiveresponses to Dragendorff and Rydon-Smith reagents, but negativeresponses to Erhlich, Sakaguchi and ninhydrin reagents. UV absorptionmaxima were observed at about 219, 242 and 317 nm in methanol and acidicmethanol which shifted to about 221, 252, 293 and 342 nm in alkalinemethanolic solution. The IR spectra of these compounds exhibited incommon the presence of amide at 1660-1680 cm⁻¹. The IR spectra forBU-3557B₁ and BU-3557C4 showed an additional carbonyl absorption at 1705cm⁻¹ which is attributed to the ketone carbonyl group in the alkyl chainof R.

As indicated, the structure of BU-3557B₂ was elucidated by x-raycrystallography. The spectral data was consistent. The ¹ H-NMR spectrumindicated one C-CH3 (δ: 1.18, 3H, d), three CH₂ (1.64, 2H, 2.00 and2.20, 2H and 3.00 and 3.25, 2H), one CH (3.94, 1H two OCH₃ (3.97, 3H and4.09, 3H) and one aromatic proton (7.90, 1H). The spectrum alsocontained four other protons (5.62, 6.88, 9.78, and 12.91) whichdisappeared upon D₂ O addition. The ¹³ C-NMR displayed seven aliphaticcarbon signals at δ: 22 ), 23.3 (q), 31.7 (t), 39.0 (t), 60.7 (q), 60.8(q) and 65.2 . (d) in addition to eight aromatic carbon signals and onecarbonyl signal. The ¹³ C- and ¹ H-NMR studies demonstrated that theother components possessed an identical bis-heteroaromatic nucleus withthat of BU-3557B₂, while differing only in the alkyl side chain.

A complex comprising the specifically recited compounds (BU-3557A₁,BU-3557A₂, BU-3557A₃, BU-3557B₁, BU-3557B₂, BU-3557B₃, BU-3557C₁,BU-3557C₂, BU-3557C₃, BU-3557C₄, BU-3557C₅, and BU-3557D) is prepared byfermenting a culture of a newly uncovered strain of Saccharothrix and isreadily recovered, e.g., by adsorption utilizing nonionic porous polymerresin absorbent. The individual compounds can be recovered from thecomplex by chromatography.

The newly uncovered strain of Saccharothrix has been designatedSaccharothrix aerocolonigenes strain N806-4. This organism was isolatedfrom a soil sample collected in Sankishwar, Karnataka State, India. Abiologically pure culture of this organism has been deposited with theAmerican Type Culture Collection, 12301 Parklawn Drive, Rockville, Md.,20852, and added to its permanent collection of microorganisms as ATCC53712. The permanency of the deposit of this culture and readyaccessibility thereto by the public are afforded throughout theeffective like of the patent in the event the patent is granted. Accessto the culture is available during the pendency of the application under37 CFR 1.14 and 35 USC 112. All restrictions on the availability to thepublic of the culture deposited will be irrevocably removed upongranting of the patent.

The morphology, cultural and physiological characteristics, and cellchemistry of strain N806-4 indicate genus Saccharothrix. Based on thefurther physiological characterization, the strain was identified as S.aerocolonigenes. We turn now in detail to the morphology, culturalcharacteristics, physiologic characteristics, cell chemistry andtaxonomic position.

Morphology Strain N806-4 forms branched hyphae which develop intosubstrate and aerial mycelia. Typical nocardioform fragmentation ofsubstrate mycelium, which affords rod or coccoid elements, is notobserved, but partial fragmentation occurs. Lone straight chains ofcylindrical segments (0.4-0.6×0.8-1.8 μm) are formed in the total partsof aerial mycelium on tyrosine agar (=ISP medium No. 7) orglucose-asparagine agar. After further incubation, spores are formeddiscontinuously with intercalary empty hyphae in the segmented aerialmycelium. The mature spores are oval (0.6-0.8×0.8-1.2 μm), non-motile,and have smooth surfaces. The aerial mycelia often fuse into thickfascicle which occurs also in the sporulated hyphae. Constricted zigzaghyphae and sclerotic granule are occasionally observed on the aerialmycelium.

Cultural characteristics: Aerial mycelium is formed moderately onglycerol-asparagine agar (=ISP medium No. 5) and tyrosine agar (=ISPmedium No. 7), and very poorly or not formed in ISP media Nos. 2, 3, 4,and 6 and Czapek's sucrose-nitrate agar. The color of the aerialmycelium is white. The color of the substrate mycelium is colorless,orange yellow or light yellowish brown. Melanoid and other diffusiblepigments are not formed. The cultural characteristics are presented inTable IV below.

                                      TABLE IV                                    __________________________________________________________________________    Cultural characteristics of strain N806-4                                                    Growth                                                         Medium         Aerial mycelium                                                                           Substrate mycelium                                                                          Diffusible pigment                   __________________________________________________________________________    Sucrose nitrate agar                                                                         Moderate    Moderate orange yellow (71)                                                                 Pale yellow (89)                     (Czapek-Dox agar)                                                                            Scant; white                                                   Tryptone-yeast extract broth                                                                 Moderate, floccose and    None                                 (ISP No. 1)    not turbid                                                     Yeast extract-malt extract agar                                                              Good        Dark orange yellow (72)                                                                     None                                 (ISP No. 2)    Poor; white                                                    Oat meal agar (ISP No. 3)                                                                    Moderate    Moderate yellow (87) to                                                                     None                                                Poor; white colorless                                          Inorganic salts-starch agar                                                                  Moderate    Moderate orange yellow (71)                                                                 None                                 (ISP No. 4)    Poor; white to light orange yellow (70)                        Glycerol-asparagine agar                                                                     Moderate    Pale yellow (89)                                                                            None                                 (ISP No. 5)    Moderate; white                                                Peptone-yeast extract-iron agar                                                              Moderate    Colorless     None                                 (ISP No. 6)    Scant; white                                                   Tyrosine agar (ISP No. 7)                                                                    Moderate    Moderate olive brown (95)                                                                   Grayish yellow (90)                                 Moderate; white                                                Glucose-asparagine agar                                                                      Moderate    Pale yellow (89)                                                                            None                                                Poor; white                                                    Nutrient agar  Poor        Colorless     None                                                None                                                           Bennett's agar Moderate    Dark orange yellow (72)                                                                     None                                                Poor; white                                                    Papavizas' V-8 juice-dextrose-                                                               Moderate    Strong yellowish brown (74)                                                                 None                                 yeast extract agar                                                                           Poor; white                                                    __________________________________________________________________________     Observation after incubation at 28° C. for 3 weeks.                    Color and number in parenthesis follow IS0CNBC designation.              

Physiological characteristics' The growth temperature ranges from 22° C.to 42° C. No growth occurs at 18° C. and 45° C. The growth is restrictedby NaCl at 5% but not at 4% or less. It is resistant to lysozyme. Thephysiological characteristics and carbohydrate utilization are presentedin Table V below. The results of additional physiological test (asdescribed by Gordon, R. E. et al, 37 Some bits and pieces of the genusNocardia: N. carnea. N. vaccinii, N. transvalensis. N. orientalis and N.aerocolonigenes", J. Gen. Microbiol. 109: 69-78, 1978) are presented inTable VI below.

                  TABLE V                                                         ______________________________________                                        Physiological characteristics of strain N806-4                                ______________________________________                                        Hydrolysis of:                                                                Gelatin              +                                                        Starch:                                                                       Soluble starch       +                                                        Potato starch        +(w)*                                                    Milk coagulation     +                                                        Peptonization        +                                                        Production of:                                                                Nitrate reductase    +                                                        Tyrosinase           -                                                        Tolerance to:                                                                 Lysozyme, 0.01% (w/v)                                                                              +                                                        NaCl, 1˜4% (w/v)                                                                             +                                                        NaCl, 5% (w/v)       -                                                        Temperature:                                                                  Growth range         22° C.˜42° C.                        No growth            18° C. & 45° C.                            pH:                                                                           Growth               4.5˜12.0                                           No growth            4.0 & 12.5                                               Utilization of:                                                               Glycerol             +                                                        D(-)-Arabinose       +(w)                                                     L(+)-Arabinose       +                                                        D-Xylose             +                                                        D-Ribose             +                                                        L-Rhamnose           +(w)                                                     D-Glucose            +                                                        D-Galactose          +                                                        D-Fructose           +                                                        D-Mannose            +                                                        L(-)-Sorbose         -                                                        Sucrose              +                                                        Lactose              +                                                        Cellobiose           +                                                        Melibiose            +                                                        Trehalose            +                                                        Raffinose            +(w)                                                     D(+)-Melezitose      -                                                        Soluble starch       +                                                        Cellulose            -                                                        Dulcitol             -                                                        Inositol             +                                                        D-Mannitol           +                                                        D-Sorbitol           -                                                        Salicin              +                                                        ______________________________________                                         *+(w): weakly positive                                                   

                  TABLE VI                                                        ______________________________________                                        Additional physiological characteristics of strain N806-4                     ______________________________________                                        Hydrolysis of:                                                                Adenine             -                                                         Hypoxanthine        +                                                         Tyrosine            +                                                         Xanthine            -                                                         Casein              +                                                         Urea                +                                                         Esculin             +                                                         Hippuric acid       +                                                         Survival at 50° C., 8 hr                                                                   -                                                         Utilization of:                                                               Benzoate            -                                                         Citrate             +                                                         Mucate              -                                                         Succinate           +                                                         Tartrate            -                                                         Acid formation:                                                               Glycerol            +                                                         D(-)-Arabinose      +                                                         L(+)-Arabinose      +                                                         D-Xylose            +                                                         L-Rhamnose          +                                                         D-Glucose           +                                                         D-Mannose           +                                                         Lactose             +                                                         Cellobiose          +                                                         Melibiose           +                                                         Trehalose           +                                                         Raffinose           +                                                         D(+)-Melezitose     -                                                         Inositol            +                                                         D-Mannitol          +                                                         D-Sorbitol          -                                                         Erythritol          -                                                         Methyl α-glucoside                                                                          -                                                         Adonitol            -                                                         ______________________________________                                    

Cell chemistry: The presence of meso-diaminopimelic acid, ribose,glucose, galactose and mannose in the whole cell hydrolysate of strainN806-4 indicates that the strain belongs to the cell wall type III andsugar pattern C. The strain has the type PII phospholipid (presence ofphosphatidylethanolamine and phosphatidylinositol), and containsMK-9(H₄) as the major menaquinone. Glycolate test is negative. Mycolicacid is absent.

Taxonomic position: The morphology, cultural characteristics and cellchemistry of strain N806-4 place it in the genus Saccharothrix, (Labeda,D. P. et al, "Saccharothrix: a new genus of the Actinomvcetales relatedto Nocardiopsis, "Int. J. Syst. Bacteriol 34' 426-431, 1984) in whichtwo species, S. australiensis (See Labeda, D. P. et al infra.) and S.aerocolonigenes (Labeda, D., Int. J. System. Bacteriol. 36: 109-110,1986, and Shinobu, R., et al, Bot. Mag, Tokyo, 73: 212-216, 1960) havebeen described. S. australiensis is different from strain N806-4 in thefollowing characteristics: the formation of light brownish gray aerialmycelium and dark brown vegetative mycelium in ISP medium No. 2, andclear difference in Gordon's physiological tests. Among the knownstrains of S. aerocolonigenes. only strain C38383 (ATCC 39243) which isa rebeccamycin-producing organism, forms spore chains on the aerialmycelium. Morphologically, strain N806-4 is related to strain C38383. Inaddition, strain N806-4 is well consistent with strain C38383 and otherstrains of S. aerocolonigenes in the physiological characteristics ofGordon's tests. Thus strain N806-4 was classified as Saccharothrixaerocolonigenes.

The fermentation of a culture of the newly uncovered strain ofSaccharothrix which has been designated Saccharothrix aerocolonigenesstrain N806-4 (ATCC 53712) or variant or mutant thereof to produce theaforedescribed antifungal antibiotic complex (comprising BU-3557A₁,BU-3557A₂, BU-3557A₃, BU-3557B₁, BU-3557B₂, BU-3557B₃, BU-3557C₁,BU-3557C₂, BU-3557C₃, BU-3557C₄, BU-3557C₅ and BU-3557D), which issometimes referred to hereafter as BU-3557 complex, is carried out underaerobic conditions in an aqueous nutrient medium containing assimilablesources of carbon and nitrogen until a substantial amount of saidcomplex is produced, i.e., an amount suitable for recovery. In theproduction of novel antifungal antibiotic compound here, BU-3557 complexis recovered from the fermentation medium and individual compounds areseparated from the complex, e.g., by chromatography. Other compoundswithin the genus herein are prepared from said individual compounds, ifdesired, by methods known for substituting on alkyl substituent at the2-position of thiazolyl moiety or rearranging substituents thereon, withappropriate blocking groups being utilized if necessary.

A biologically pure culture of the microorganism Saccharothrixaerocolonigenes strain N806-4 (ATCC 53712) which is capable of producingsaid complex in a recoverable quantity upon aerobic cultivation is anaqueous nutrient medium containing assimilable sources of carbon andnitrogen is novel and constitutes one aspect of the invention herein.

Turning now to the aforedescribed fermentation, the aqueous nutrientmedium consists essentially of assimilable sources of carbon andnitrogen and sterile water. Calcium source (very preferably calciumcarbonate) is preferably present. Sodium chloride can also be included.Typically, carbon source is present in an amount by weight of from about0.5% to about 5%, and nitrogen source is present in an amount by weightof about 0.2% to about 5%. Calcium source is preferably present in anamount by weight ranging from 0.05% to about 1%. Sodium chloride can beincluded, for example, in an amount of about 0.1% to about 0.3% byweight.

Suitable carbon sources include, for example, arabinose, xylose, ribose,glucose, fructose, sucrose, lactose, soluble starch, mashed potatoes,glycerol, mannitol, sorbitol, etc.

Suitable nitrogen sources include, for example, casein, NZ-case, soybeanmeal, soy flour, soybean powder, cottonseed meal, cottonseed flour,peanut meal, wheat gluten, meat meal, meat, fish meal, fish extract,yeast extract, peptone, corn steep liquor, etc.

The fermentation is effected at any temperature conducive tosatisfactory growth of the producing organism, i.e., 22° C. to 42° C.and is conveniently carried out at a temperature of about 27° C. to 32°C. Preferably, the fermentation is carried out under submerged aerobicconditions with agitation.

In carrying out said fermentation, preferably a see culture obtained bygrowing from a slant is used to inoculate either shake flasks orinoculum tanks. The seed culture is readily produced by shakecultivation for from 3 to 5 days. Cultivation in the shake flasks orinoculum tanks is preferably carried out until assay indicates peakantibiotic (antifungal potency determined, for example, using anindicator organism (e.g., Candida alibcans A9540) or HPLC.

A shake flask is usually run at 100 to 300 rpm and normally reachesmaximum growth in 5 to 7 days. An inoculum tank may be agitated at 100to 300 rpm and is preferably employed with aeration rate of 1/2 to 2volumes of sterile free air per volume of fermentation broth per minuteforced into the broth through a sparger and under these conditions peakantibiotic potency is normally obtained in 3 to 5 days.

The produced antibiotic complex is readily recovered by harvesting theresulting fermentation broth, separating the broth to cake andsupernatant, e.g., utilizing a centrifuge, and recovering the antibioticcomplex from the supernatant, for example, by absorbing it utilizingnonionic porous (macroreticular) polymer resin, e.g., Diaion HP-20 fromMitsubishi Kasei, and eluting the complex therefrom and concentrating ina plurality of stages to recover a crude solid comprising the antibioticcomplex which is separated into individual antibiotic components withinthe scope of the invention by chromatography.

As indicated hereinbefore the compounds of the instant invention areuseful for controlling fungal infections and are preferably administeredin the form of a pharmaceutical composition comprising an effectiveantifungal amount of compound of the instant invention in combinationwith a pharmaceutically acceptable carrier or diluent.

Antifungal compositions herein may be made up in any suitable formappropriate for desired use, e.g., oral, parenteral or topicaladministration. Examples of parenteral administration are intramuscularintravenous, intraperitoneal, rectal and subcutaneous administration.

The diluent or carrier ingredients should not be such as to diminish thetherapeutic effects of the antifungal antibiotic.

Suitable dosage forms for oral use include tablets, dispersible powders,granules, capsules, suspensions, syrups, and elixers. Inert diluents andcarriers for tablets include, for example, calcium carbonate, sodiumcarbonate, lactose and talc. Tablets may also contain granulating anddisintegrating agents such as starch and alginic acid, binding agentssuch as starch, gelatin and acacia, and lubricating agents such asmagnesium stearate, stearic acid and talc. Tablets may be uncoated ormay be coated by known techniques, e.g., to delay disintegration andabsorption. Inert diluents and carriers which may be used in capsulesinclude, for example, calcium carbonate, calcium phosphate and koalin.Suspensions, syrups and elixers may contain conventional excipients, forexample, methyl cellulose, tragacanth, sodium alginate; wetting agents,such as lecithin and polyoxyethylene stearate; and preservatives, e.g.,ethyl-p-hydroxybenzoate.

Turning now to dosage forms suitable for parenteral administration,these include solutions, suspensions, dispersions, emulsions and thelike. They may also be manufactured in the form of sterile solidcompositions which can be dissolved or suspended in sterile injectablemedium immediately before use. They may contain suspending or dispersingagents known in the art.

For topical application, pharmaceutical compositions containing compoundwithin the scope of the present invention in combination with apharmaceutical carrier or diluent may be in the form, for example, ofsolutions, pastes, creams, ointments, gels and waxes. Suitable carrierand diluent ingredients include, for example, zinc oxide paste, sorbitolsolution, water, starch, mineral oil, white petrolatum, waxes (e.g.,paraffin), fatty alcohols (e.g. cetyl alcohol) and fatty acid esters(e.g., containing 12 to 18 carbon atoms in the fatty acid moiety and 1to 3 carbon atoms in the ester moiety, such as isopropyl laurate,isopropyl myristate, isopropyl palmitate, methyl stearate or ethylstearate) and fatty oils.

One aspect of the invention herein is directed to therapeuticallytreating on animal host affected by a fungal infection sensitive tocompound of the instant invention which comprises administering to saidhost an effective antifungal dose of said compound. It will beappreciated that the actual preferred amount of compound of the instantinvention used will vary according to the particular compound, theparticular composition formulated, the mode of application and theparticular situs, host and disease being treated. Many factors thatmodify the action will be taken into account by those skilled in theart, e.g., age, body weight, sex, diet, time of administration, route ofadministration, rate of excretion, condition of the host, drugcombinations, reaction sensitivities and severities and severity ofdisease. Administration can be carried out continuously or periodicallywithin the maximum tolerated dose. Optimal application rates for a givenset of conditions can be ascertained by those skilled in the art usingconventional dosage administration tests in view of the aboveguidelines.

Compounds within the present invention have exhibited significant invitro antifungal activity against Candida alibcans. Aspergillusfumiqatus and Trichophvton mentagrophvtes by the serial broth dilutionmethod and moderate in vitro antifungal activity against Candidaalbicans, Cryptococcus neoformans. Aspergillus fumigatus. Piriculariaoryzae. Trichophyton mentagrophytes, Blastomyces dermititidis.Sporothrix schenckii and Mucor spinosus. Furthermore, in vivo antifungalactivity against C. albicans systemic infection has been indicated atdoses of 3.1 to 25 mg/kg, preferably 6.3 to 12.5 mg/kg for compounds ofthe invention where R is 1,5-dihydroxy-5,5-dimethylpentyl (BU-3557A₃)when administered intramuscularly twice a day for two days. Furthermore,in vivo antifungal activity against Candida albicans vaginal infectionhas been demonstrated for BU-3557B₂ when administered topically for 5days at an EC₅₀ (concentration for 50% inhibition compared to a control)of 0.34%.

In vitro antibacterial activity has also been demonstrated againstGram-positive bacteria for compounds of the invention herein.

Furthermore BU-3557B₂ has demonstrated in vitro antiprotazoal(anti-trichomonal) activity against a clinical isolate of trichomonasvaginalis.

The following examples are provided for illustrative purposes only andare not intended to limit the scope of the invention.

EXAMPLE I Production of Antifungal Complex BU-3557 By Flask Fermentation

A loopful of the slant culture of Saccharothrix aerocolonigenes strainN806-4 (ATCC 53712) was inoculated in a 500-ml Erlenmeyer flaskcontaining 100 ml of the following seed medium' soluble starch 0.5%,glucose 0.5%, fish extract (Mikuni) 0.1%, yeast extract (Oriental) 0.1%NZ-case (Sheffield) 0.2% and CaCO₃ 0.1%. Shake cultivation was carriedout at 28° C. for 4 days on a rotary shaker (200 rpm). The seed culture(2 ml) was inoculated into a 500-ml Erlenmeyer flask which contained 100ml of the fermentation medium composed of mashed potato (Snow MilkProducts) 4% corn steep liquor 2%, NaCl 0.2% and CaCO₃ 0.3%; beingadjusted to pH of 7.2 before being autoclaved. The fermentation wascarried out on a rotary shaker (200 rpm) at 28° C. for 8 days. For theassay of antifungal activity, the broth dilution method was used with a96 -well microtitre plate using Candida alibcans A9540 as the indicatororganism. The activity was determined by Titertek Multiskan MCC (FlowLaboratories) at 540 nm. HLPC was also used for determining BU-3557complex. The antifungal activity of the flask fermentation reachedmaximum on the 6th day.

EXAMPLE II Production of Antifungal Complex BU-3557 By Tank Fermentationand Separation of Complex into Components

(1) Fermentation to produce BU-3557 complex

Tank fermentation was carried out. The seed culture was prepared by thesame procedure as in Example I using the following seed medium: glucose2%, soybean meal 1% and CaCO₃ 0.5%. After shaking cultivation on arotary shaker at 28° C. for 4 days, the seed culture (2 liters) wasinoculated to 200-liter fermentation tank (Marubishi MPF-U200) whichcontained 120 liters of the fermentation medium having the samecomposition as for the flask fermentation in Example I. The tankfermentor was operated at 28° C. with agitation at 250 rpm and aerationrate of 120 liters/min. The broth pH gradually rose with the progress offermentation and reached 7.7 to 7.8 after 90 hrs when a peak antibioticpotency of 112 μg/ml (HPLC assay) was obtained.

(2) Isolation and purification of components of BU-3557 complex

(a) Extraction and primary separation

The harvested fermentation broth (265L) was separated to supernatant andmycelial cake by use of a Sharpless A centrifuge (Kokusan No. 4A). Theclear supernatant (232 L) was stirred wit Diaion HP-20 (23 L) for 1.5hours. (Diaion HP-20 is a trademark of Mitsubishi Chemical Industries ofJapan for a nonionic macroreticular, i.e., macroporous, polymer resin).The resin was collected by filtration, washed twice with 50% aqueousmethanol (22 L each) and eluted three times with 23 L each of 80%aqueous acetone. The eluates were combined and concentrated in vacuo andthe aqueous concentrate (17 L) was charged on a column of Diaion HP-20(3.4 L). The column was washed with 50% aqueous methanol (10 L) anddeveloped with 60% aqueous acetone. The eluate was collected infractions which were examined by paper-disc assay using Cryptococcusneoformans IAM-4514 (Cn-2). The active fractions were pooled,concentrated in vacuo and extracted twice with 3.5 L each of ethylacetate. Evaporation of the extract in vacuo yielded a crude solid ofBU-3557 complex (18.5 g).

Crude solid (2.4g) was applied on a column of LiChroprep RP-18 (Merck,22×450 mm) which had been equilibrated by a mixture of acetonitrile and0.15% KH₂ PO₄ (36:64 v/v, pH 3.5 by an addition of H₃ PO₄). Elution wascarried out with the same solvent mixture using an FMI A medium pressureLAB pump (Fluid Metering Inc.), and the eluate was monitored by Cn-2 andHPLC. BU-3557A complex was eluted first followed by BU-3557B₂, then amixture of BU-3557B₁, B₂ and B₃ complex and finally BU-3557C and Dcomplex. Each fraction was concentrated in vacuo to remove theacetonitrile and extracted with ethyl acetate. The extract wasconcentrated in vacuo to give the four fractions of BU-3557. Repetitionof this chromotography seven times starting from 2.4 g each of crudesolid yielded BU-3557A complex (2.4g), semi-pure BU-3557 B₂ (3.4g),BU-3557B₁, B₂ and B₃ complex (2.Og) and BU-3557C and D complex (1.3 g).

(b) Purification of BU-3557A complex

The BU-3557A complex obtained above was a mixture of threesub-components (BU-3557A₁, A₂, A₃) by HPLC analysis. The complex solid(2.3 g) was chromatographed on a column of LiChroprep RP-18 (22×450 mm)eluting with acetonitrile-0.15% KH₂ PO₄, pH 3.5 (33.3: 66.7 v/v)mixture. Upon monitoring the eluate by HPLC, BU-3557A₁, and A2 complexwas eluted first followed by nearly only BU-3557A₃. The relevantfractions were concentrated and each concentrate was extracted withethyl acetate. Evaporation of the extracts gave semi-pure BU-3557A₁ andA₂ complex (601 mg) and semi-pure BU-3557A₃ (984 mg).

The BU-3557A₁ and A₂ complex (550 mg) was re-chromatographed on a columnof YMC GEL ODS A60 (Yamamura Chemical Co., 22×450 mm). Elution wascarried out with a mixture of methanol-0.03 M phosphate buffer pH 7.0(48:52, v/v) and the bioactive eluates were examined by HPLC. The firstactive fractions containing homogeneous BU-3557A₁ were pooled,concentrated and then extracted with ethyl acetate. Evaporation of theextract yielded a white solid of pure BU-3557A₁ (41 mg). The secondactive fractions were similarly worked up to give pure BU-3557A₂ (18mg).

The semi-pure sample of BU-3557A₃ (705 mg) was purified by a column ofYMC GEL ODS A60 (22×450 mm) developed with a mixture of methanol-0.03Mphosphate buffer pH 7.0 (50:50, v/v). Upon examination of the eluates byHPLC, the heart-cuts of BU-3557A₃ fractions were concentrated in vacuoto remove the methanol. Extraction of the concentrate with ethyl acetatefollowed by concentration of the extract afforded pure BU-3557A₃ (123mg).

(c) Purification of BU-3557B fraction

The BU-3557B₂ main fraction (568 mg) was purified by a preparative HPLCusing a Gilson Model 303 system with a semi-preparative SSC-ODS-842column (Senshu Kagaku Co. 30×250 mm). The elution solvent was a mixtureof methanol-0.03M phosphate buffer pH 7.0 (Time 0-15 min. 54/46, 15-80min.: linear gradient of 54/46-65/35 and 80-100 min.: 65/35 v/v, flowrate 12.0 ml/min) and eluates were monitored by UV absorption at 280 nm.BU-3557B₁ was eluted at the retention time of 47 min. followed byBU-3557B₂ at 58 min. and then BU-3557B₃ at 72 min. The appropriateeluates were pooled and concentrated to remove the methanol. The aqueousconcentrate of BU-3557B₁ fractions were passed through a column ofDiaion HP-20 (20×300 mm). The column was washed with 50% aqueousmethanol (200 ml) and then eluated with 80% aqueous acetone (100 ml).Evaporation of the acetone eluate yielded homogeneous BU-3557B₁ (22.3mg). The concentrates of BU-3557B₂ and B₃ fractions were similarlytreated to give pure solids of BU-3557B₂ (229.6 mg) and BU-3557B₃ (15.4mg) respectively. The crude solid of BU-3557 B₁, B₂, B₃ complex (750 mg)was purified by the preparative HPLC as described above to yield pureBU-3557B₁ (54.4 mg), B₂ (55.3 mg) and B₃ (203.9 mg).

(d) Purification of BU-3557C and D complex

Separation of BU-3557C and D complex (1.21 g) was achieved by a mediumpressure liquid chromotography with a LiChroprep RP-18 column (22×450mm). The elution was performed first with a mixture of methanol-0.03Mphosphate buffer pH 7.0 (52/48 v/v, 1.0 L and then a linear gradient ofthe mixture 52/48 to 60/40, a total volume 1.OL). Fractionation of theeluate was guided by HPLC and the two active fractions were worked up toyield BU-3557C complex (545 mg) and pure BU-3557D (379 mg).

BU-3557C complex contained five sub-components, BU-3557C₁, C₂, C₃, C₄,C₅ by HPLC analysis. The complex (66 mg) was charged on asemi-preparative SSC-ODS-842 column (30×250 mm). Elution was carried outwith a mixture of acetonitrile and 0.15% KH pH 3.5 (Time 0-15 min.:25/75-41.5/58.5, 15-70 min: 41.5/58.5, 70-90 min.: 41.5/58.5-55/45).Upon monitoring by UV absorption at 300 nm, the relevant fractions (C₁ :Rt 57.2, C₂ 58.5, C₃ : 63.2, C_(4:) 70.2 and C₅ : 80.7) were pooled.This semi-preparative HPLC was repeated 7 times and the appropriatefractions were combined. Each fraction was concentrated and extractedtwice with ethyl acetate. Evaporation of the extracts gave pureBU-3557C₁ (22.8 mg), BU-3557C₂ (23.6 mg), BU-3557C₃ (110.0 mg),BU-3557C₄ (75.0 mg) and BU- 3557C₅ (33.0 mg).

EXAMPLE III

BU-3557 complex is prepared in a similar manner so that prepared inExample I.

HPLC of the components of said complex is shown in Table VII below.

                  TABLE VII                                                       ______________________________________                                        Component     Retention time                                                  ______________________________________                                        BU-3557 A.sub.1                                                                             7.14                                                            BU-3557 A.sub.2                                                                             7.46                                                            BU-3557 A.sub.3                                                                             7.89                                                            BU-3557 B.sub.1                                                                             9.44                                                            BU-3557 B.sub.2                                                                             8.87                                                            BU-3557 B.sub.3                                                                             9.57                                                            BU-3557 C.sub.1                                                                             10.34                                                           BU-3557 C.sub.2                                                                             10.63                                                           BU-3557 C.sub.3                                                                             10.99                                                           BU-3557 C.sub.4                                                                             11.53                                                           BU-3557 C.sub.5                                                                             12.48                                                           BU-3557 D     11.72                                                           ______________________________________                                        Condition                                                                     column: Microsorb Short One C.sub.18 (4.6 mm I.D. × 100 mm, 3           μm)                                                                        mobile phase: Solvent A: 0.15% KH.sub.2 PO.sub.4 adjusted to pH 3.5 with      H.sub.3 PO.sub.4                                                              Solvent B: CH.sub.3 CN                                                        Time (min.)                                                                           0-3            3-9     9-14                                           A/B     85/15-60/40    60/40   60/40-45/55                                    flow rate: 1.2 ml/min                                                         detection: UV absorption at 254 nm                                        

EXAMPLE IV In Vitro Antifungal Activity

The in vitro antifungal activity of the components of BU-3557 wasdetermined against various fungi by serial broth (results in Table VIIIbelow) and agar (results in Table IX below) dilution methods. Sabourauddextrose broth and agar were used for these experiments. The inoculumsize was adjusted to 10⁴ -10⁶ and 10⁵ -10⁹ CFU/ml for both methods.After incubation at 28° C. for 48 or 72 hours, the minimum inhibitoryconcentration (MIC), the lowest concentration of antibiotic causingvirtually complete inhibition of growth, was examined. As shown in TableVIII, all components of BU-3557 exhibited significant in vitroantifungal activity against Candida albicans. Aspergillus fumigatus andTrichophyton mentagrophytes in the broth dilution method. As shown inTable IX, all components of BU-3557 exhibited moderate antifungalactivity against various clinically important pathogenic fungi inSabouraud dextrose agar.

                                      TABLE VIII                                  __________________________________________________________________________    In vitro antifungal activity in Sabouraud dextrose broth                                       MIC (μg/ml).sup.1                                                          BU-3557                                                      Test organism.sup.2                                                                            A1  A2  A3  B1 B2                                                                              B3 C1 C2 C3 C4 C5 D                         __________________________________________________________________________    Candida albicans A9540                                                                         50  >100                                                                              >100                                                                              12.5                                                                             3.1                                                                             6.3                                                                              3.1                                                                              3.1                                                                              6.3                                                                              6.3                                                                              6.3                                                                              3.1                                        (1.6).sup.3                                                                       (3.1)                                                                             (3.1)       (1.6)                                                                            (1.6)                                                                            (3.1)                                                                            (3.1)                                                                            (3.1)                        Aspergillus fumigatis IAM 2034                                                                 6.3 12.5                                                                              6.3 6.3                                                                              6.3                                                                             12.5                                                                             3.1                                                                              3.1                                                                              3.1                                                                              3.1                                                                              3.1                                                                              3.1                                        (3.1)                                                                             (6.3)                                                                             (3.1)                                                Trichophyton mentagrophytes #4329                                                              >100                                                                              >100                                                                              >100                                                                              6.3                                                                              6.3                                                                             6.3                                                                              6.3                                                                              6.3                                                                              6.3                                                                              6.3                                                                              6.3                                                                              6.3                                        (25)                                                                              (50)                                                                              (50)        (3.1)                                                                            (3.1)                                                                            (3.1)                                                                            (3.1) (3.1)                     __________________________________________________________________________     .sup.1 Determined after incubation for 48-72 hours at 28° C.           .sup.2 Inoculum size: 10.sup.4 -10.sup.6 CFU/ml                               .sup.3 Values in parenthesis indicate partial inhibition                 

                                      TABLE IX                                    __________________________________________________________________________    In vitro antifungal activity in Sabouraud dextrose agar                                         MIC (μg/ml).sup.1                                                          BU-3557                                                     Test organism.sup.2                                                                             A1  A2  A3  B1 B2 B3 C1  C2  C3  C4  C5  D                  __________________________________________________________________________    Candida albicans IAM 4888                                                                       25  100 >100                                                                              50 25 50 50  50  50  50  50  50                                   (12.5).sup.3                                                                      (12.5)                                                                            (<6.3)       (12.5)                                                                            (<6.3)                                                                            (<6.3)                                                                            (12.5)                                                                            (12.5)                                                                            (12.5)             Candida albicans A 9540                                                                         25  >100                                                                              >100                                                                              25 25 50 50  50  50  50  50  50                                   (12.5)                                                                            (12.5)                                                                            (<6.3)       (<6.3)                                                                            (<6.3)                                                                            (<6.3)                                                                            (12.5)                                                                            (<6.3)                                                                            (12.5)             Cryptococcus neoformans D 49                                                                    25  >100                                                                              >100                                                                              50 25 50 50  50  50  50  50  50                 Cryptococcus neoformans IAM 4514                                                                25  >100                                                                              >100                                                                              25 25 50 50  50  50  50  50  50                 Aspergillus fumigatus IAM 2530                                                                  25  25  25  50 25 50 25  25  25  25  25  25                 Aspergillus fumigatus IAM 2034                                                                  25  25  25  50 25 50 25  25  25  25  25  25                 Piricularia oryzae D 91                                                                         50  50  25  NT.sup.4                                                                         NT NT 25  25  25  25  25  25                 Trichophyton mentagrophytes D155                                                                100 50  50  NT 25 NT 25  25  25  25  25  25                 Trichophyton mentagrophytes #4329                                                               100 50  50  50 25 50 25  12.5                                                                              25  25  25  25                 Blastomyces dermatitidis D 40                                                                   NT  25  25  12.5                                                                             25 25 12.5                                                                              12.5                                                                              12.5                                                                              25  12.5                                                                              25                 Sporothrix schenckii IFO 8158                                                                   12.5                                                                              >100                                                                              >100                                                                              50 50 50 25  25  25  25  25  25                 Mucor spinosus IFO 5317                                                                         6.3 12.5                                                                              12.5                                                                              25 12.5                                                                             25 <6.3                                                                              <6.3                                                                              <6.3                                                                              12.5                                                                              <6.3                                                                              <6.3               __________________________________________________________________________     .sup.1 Determined after incubation for 72 hours at 28° C.              .sup.2 Inoculum size: 10.sup.5 -10.sup.9 CFU/ml                               .sup.3 Values in parenthesis indicate partial inhibition                      .sup.4 Not tested                                                        

EXAMPLE V In vivo Antifungal Activity of BU-3557A₃ Against

C. albicans Systemic Infection

Testing for In vivo therapeutic efficacy of BU-3557A3 (60% purematerial) against C. albicans A9540 intravenous infection was carriedout. The test organism was cultured for 18 hours at 28° C. in YGP medium(yeast extract, glucose, peptone, K₂ HPO₄, MgSO₄) and suspended insaline. Male ICR mice weighing 20 to 24 g were infected intravenouslywith about 10 time the median lethal dose of the fungi. BU-3557A₃ wasadministered to groups of 5 mice intramuscularly twice a day on days 0and 1. The results are set forth in Table X below. The dose thatprotects 50% of the animals from the infection m(PD₅₀, mg/kg) wascalculated from survival rates examined on the 20th day after infection.BU-3557A₃ demonstrated good in vivo efficacy, though the highest dose at25 mg/kg showed weak toxic sign.

                  TABLE X                                                         ______________________________________                                        In vivo antifungal activity against Candida albicans                          A 9540 intravenous infection in mice                                                   Dose                                                                 Compound (mg/kg, im/inj.).sup.1                                                                      Number of survivors/tested                             ______________________________________                                        Bu-3557 A3                                                                             25            1/5                                                    (purity 60%)                                                                           12.5          3/5                                                             6.3           3/5                                                             3.1           1/5                                                    ______________________________________                                         PD.sub.50 (mg/kg, im/inj.): 5.4                                               .sup.1 Compound was administered intramuscularly twice a day on days 0 an     1.                                                                       

EXAMPLE VI In Vivo Antifungal Activity of BU-3557B₂ Against

C. albicans Vaginal Infection

Topical application of BU-3557B₂ against C. albicans A9540 vaginalinfection was examined. Groups of 5 female mice (ICR, 18-21 g) weretreated subcutaneously with 0.5 estradiol benzoate 3 days before and 4days after C. albicans A9540 vaginal infection, respectively. A 0.01 mlof the cell suspension of C. albicans containing 10⁸ CFU/ml wasinoculated intravaginally on day 0. Then 0.02 ml of the compoundsolution in Solbase (1'1 mixture of polyethyleneglycol 400 andpolyethyleneglycol 4000, Dainippon-Seiyaku) was instilled intravaginallyonce a day on days 0 to 4. On the day 7, vaginal exudate was taken by athin glass rod and spread on the YGP agar plate. Viable cell count wasmade after incubation for 2 days at 28° C. The results are shown inTable XI below. The EC₅₀ value, which is the drug concentration (weight%) giving 50% inhibition of the control, was calculated by the method ofleast squares. As shown in Table XI, BU-3557B₂ exhibited potent activityagainst C. albicans vaginal infection by topical application.

                  TABLE XI                                                        ______________________________________                                        In vivo antifungal activity against Candida albicans                          A 9540 vaginal infection in mice                                                       Conc.    No. of cells/plate                                          Compound   (%)        Mean    % inhibition                                    ______________________________________                                        Vehicle    --         284                                                     BU-3557 B2 2.0        0       100                                                        1.0        3       99                                                         0.5        10      97                                                         0.25       243     14                                                         0.13       248     13                                              ______________________________________                                         EC.sub.50 (%): 0.34                                                           Compound was applied once a day from day 0 to day 4.                     

EXAMPLE VII Testing for In Vitro Antibacterial Activity

The In vitro antibacterial activity of the components of BU-3557 wasdetermined by the serial dilution method in nutrient agar againstGram-positive and Gram-negative aerobic bacteria. The inoculum size wasadjusted to 10⁵ -10⁶ CFU/ml The MIC was defined as the lowestconcentration of test compound completely inhibiting bacterial growthafter 18 hour-incubation at 37° C. The results are shown in Table XIIbelow. As shown in Table XII, all components of BU-3557 exhibited weakin vitro antibacterial activity against Gram-positive bacteria, but werenearly inactive against Gram-negative organisms.

                                      TABLE XII                                   __________________________________________________________________________    In vitro antibacterial activity in nutrient agar                                             MIC (μg/ml).sup.1                                                          BU-3557                                                        Test organism.sup.1                                                                          A1  A2  A3  B1  B2  B3  C1  C2  C3  C4  C5  D                  __________________________________________________________________________    Staphylococcus aureus 209 P                                                                  50  50  50  25  25  50  25  50  100 25  25  50                 Staphylococcus aureus #52-34.sup.3                                                           50  100 100 50  50  100 100 100 >100                                                                              100 100 100                Staphylococcus aureus A 20239.sup.4                                                          50  100 100 50  50  100 100 100 >100                                                                              100 50  100                Staphylococcus aureus                                                                        50  100 100 50  50  100 100 100 >100                                                                              100 50  100                BX-1633-2.sup.5                                                               Staphylococcus aureus A 15097.sup.6                                                          50  50  50  25  50  50  50  100 >100                                                                              50  50  50                 Staphylococcus epidermidis                                                                   50  100 50  50  25  50  25  50  25  50  25  50                 D153                                                                          Enterococcus faecalis A 9612                                                                 100 100 100 100 100 >100                                                                              50  100 >100                                                                              100 50  50                 Bacillus subtilis PCI219                                                                     50  25  50  25  25  25  25  25  50  25  25  25                 Escherichia coli NIHJ                                                                        100 >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              100 >100                                                                              >100                                                                              100 100 100                Klebsiella pneumoniae D 11                                                                   50  >100                                                                              >100                                                                              50  50  100 >100                                                                              >100                                                                              >100                                                                              >100                                                                              100 >100               Proteus mirabilis A 9554                                                                     100 >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              100 >100                                                                              >100                                                                              100 100 100                Proteus vulgaris A 9436                                                                      100 100 100 >100                                                                              >100                                                                              >100                                                                              100 100 >100                                                                              100 100 100                Serratia marcescens A 20222                                                                  >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100               Pseudomonas aeruginosa A 9930                                                                >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100                                                                              >100               __________________________________________________________________________     .sup.1 Determined after incubation for 18 hours at 37° C.              .sup.2 Inoculum size: 10.sup.5 -10.sup.6 CFU/ml                               .sup.3 Erythromycinresistant strain                                           .sup. 4 Kanamycinresistant strain                                             .sup.5 Penicillinaseproducing strain                                          .sup.6 Methicillinresistant strain                                       

EXAMPLE VIII

Testing for In Vitro Anti-trichomonal Activity

BU-3557B₂ was tested for in vitro anti-trichomonal activity comparedwith metronidazole. Table XIII below shows MIC (minimum inhibitoryconcentration) values determined after 24 hour incubation at 37° C. Asindicated in Table XIII BU-3557B₂ exhibited appreciable activity againsta clinical isolate of Trichomonas vaginalis.

                  TABLE XIII                                                      ______________________________________                                        In vitro activity against Trichomonas                                         vaginalis in thioglycolate broth                                                            MIC (μg/ml).sup.1                                            Test organism.sup.2                                                                           BU-3557 B2  Metronidazole                                     ______________________________________                                        Trichomonas vaginalis Tv-2                                                                    25          0.8                                               ______________________________________                                         .sup.1 Determined after 24 hourincubation at 37° C.                    .sup.2 Inoculum size: 10.sup.5 cells/ml                                  

Variations will be evident to those skilled in the art. Therefore, thescope of the invention is intended to be defined by the claims.

What is claimed is:
 1. The compound having formula ##STR3## wherein R isselected from the group consisting of 1.4-dihydroxypentyl.1,4-dihydroxy-4-methylpentyl,1,5-dihydroxy-5,5-dimethylpentyl, 4-oxopentyl, 4-hydroxypentyl,4-hydroxy-4-methylpentyl, 3-hydroxypentyl, 4-methyl-5-hydroxypentyl,3-methyl-4-hydroxypentyl, 3-methyl-4-oxopentyl, 1-hydroxypentyl, and5-hydroxy-5,5-dimethylpentyl.
 2. The compound of claim 1 wherein R is4-hydroxypentyl.
 3. A pharmaceutical composition for treatment of fungalinfections comprising an effective antifungal amount of compound asrecited in claim 1 in combination with a pharmaceutical carrier ordiluent.
 4. A pharmaceutical composition as recited in claim 3 whereinsaid compound is the compound of claim
 6. 5. A method fortherapeutically treating an animal host affected by a fungal infectionsensitive to compound as recited in claim 1 which comprisesadministering to said host an effective antifungal dose of said-compound.
 6. A method as recited in claim 5 wherein said infection is aCandida albicans systemic infection and administering of said compoundis carried out parenterally.
 7. A method as recited in claim 6 whereinsaid infection is a Candida albicans vaginal infection and administeringof said compound is carried out by topical application.
 8. A method asrecited in claim 7 wherein said compound is the compound of claim 6.